The emerging knowledge on diseases and the role of natural foods in lowering the risk of such disease process have furthered research efforts for identification and development of nutritionally important dietary supplements. Milk is the first complete delivery system for essential nutrients in the newborn. This natural delivery system provides several bioactive components for critical management of gastrointestinal functions including defense against microorganisms, toxin and free radical scavenging, gut maturation and repair, nutrient diffusion and transport across mucosal barrier and prebiotic activity. Accordingly, the consumption of cow's milk has been an integral part of human civilization since antiquity and this natural delivery system has provided remarkable benefits to mankind.
Lactoferrin (LF) is a major bioactive constituent of milk. This iron-binding protein is responsible for a wide range of nutraceutical benefits and provides protection against several intestinal illnesses. LF plays an important role in various physiological pathways including inflammation by promoting neutrophil aggregation, inhibition of antibody-mediated cytotoxicity, specific growth stimulation of lymphocytes, down-regulation of myelopoiesis, complement cascade modulation by C3 convertase inhibition, intestinal iron absorption, enterocyte proliferation and gut maturation, up-regulation of thymocyte maturation, up-regulation of monocyte cytotoxicity, regulation of antibody production, regulation of cytokine production, down-regulation of tumor necrosis factor (TNF) and prevention of hydroxyradical-mediated tissue injury. Though iron chelation is considered an important molecular property of LF, a number of cellular functions are independent of this metal-binding property of LF. Specific and non-specific interactions of LF with cells, co-existence with a variety of biomolecules in different environments, molecular heterogeneity and structural flexibility confers a spectrum of multifunctionality to the LF molecule in vivo (Naidu A S, Arnold R R, Influence of lactoferrin on host-microbe interactions. In ‘Lactoferrin Interactions and Biological Functions, ed. T W Hutchens, B. Lonnerdal, pp. 259-75. Totowa, N. J., Humana Press, 1995; Naidu A S, Bidlack W R, Milk lactoferrin—Natural microbial blocking agent for food safety. Environ Nutr Interact 2:35-50, 1998).
The multifunctional activities of LF documented in several clinical trials and in vivo studies, demonstrate that this milk protein provides an excellent prebiotic physiological delivery system for the gastrointestinal tract.
Lactoferrin has been shown to modulate mucosal immunity. Oral administration of bovine LF (40 mg/day) in healthy human volunteers (n=17) results in an augmentation of their immune responses (Zimecki et al., Immunoregulatory effects of a nutritional preparation containing bovine lactoferrin taken orally by healthy individuals. Arch Immunol Ther Exp Warsz 46:231-40, 1998). Human clinical trials in Japan demonstrated a positive influence of LF consumption in primary activation of host defense. Healthy male volunteers (n=10) fed with bovine LF (2 g/day for a week) showed an improvement in their serum neutrophil function such as enhanced phagocytic activity and superoxide production by neutrophils (Yamauchi et al., Effects of orally administered bovine lactoferrin on the immune system of healthy volunteers. Adv Exp Med Biol 443:261-5, 1998). Furthermore, specific interaction of LF with alveolar macrophages, monocytes, Kupfer cells, liver endothelia, peripheral mononuclear lymphocytes, platelets, and T-lymphocytes emphasizes the role of LF in mucosal and cellular immunity.
Additionally, LF has anti-tumor activity. Activated monocytes are able to kill tumor cells and mediate antibody-dependent cell-mediated cytotoxicity. Lactoferrin markedly affects adherent monocyte toxicity, but has no effect on nonadherent lymphocytes (T-cells). Lactoferrin also enhances the natural killer (NK) activity of cells in a dose-dependent manner and augments both NK and lymphokine-activated killer (LAK) cell cytotoxic functions (Shau et al., Modulation of natural killer and lymphokine-activated killer cell cytotoxicity by lactoferrin. J Leukoc Biol 51:343-9, 1992). Lactoferrin is an effective modulator of cell-mediated immune responses, including serum cytotoxic factors, at low dosages (<1 μg/ml) however, at higher concentrations, LF-mediated induction could lead to positive or negative feedback responses, depending on the numbers and subsets of the immune cell population. Immunomodulator effects of LF, particularly the NK and LAK functions, are iron-independent, since the depletion of iron from LF by the chelator desferoxamine does not affect the cytotoxic augmentation capacity of LF. Discovery of specific LF receptors on macrophages, T- and B-lymphocytes and leukemic cells further establishes the anti-tumor potential of LF.
Lactoferrin is central to intestinal iron absorption. Research on iron absorption from milk LF has received much attention in recent years and has contributed to the development of several infant formulae (Lonnerdal B, Trace element absorption in infants as a foundation to setting upper limits for trace elements in infant formulas. J Nutr Suppl 119:1839-44, 1989). Lactoferrin has been suggested to play an important role in the intestinal absorption of iron, zinc, copper, manganese and other essential trace elements and has also been suggested to protect the gut mucosa from excess uptake of heavy metal ions. Specific LF binding receptors in the human duodenal brush border are involved in iron absorption (Cox et al., Iron-binding proteins and influx of iron across the duodenal brush border. Evidence for specific lactotransferrin receptors in the human small intestine. Biochem Biophys Acta 588:120-8, 1979). An intestinal receptor for LF (Mr 110 kDa) with a cellular density of 4.3×1014 sites per milligram of solubilized human fetal intestinal brush-border membranes (IBBM) has been isolated (Kawakami H, Lonnerdal B, Isolation and function of a receptor for human lactoferrin in human fetal intestinal brush-border membranes. Am J Physiol 261:G841-6, 1991). Furthermore, increased iron absorption via this LF receptor from IBBM during the neonatal period has been reported (Rosa G, Trugo N M, Iron uptake from lactoferrin by intestinal brush-border membrane vesicles of human neonates. Braz J Med Biol Res 27:1527-31, 1994).
The gastrointestinal tract matures most rapidly in the newborn during the period when the newborn is nursing. Human milk has been shown to stimulate thymidine uptake in a variety of fibroblast cell lines and various substances in human milk, such as epidermal growth factor, have been identified as potent mitogens. Oral administration of LF, either at low (0.05 mg/g body weight per day) or high (1 mg/g body weight per day) dosages functions as an immune stimulating factor on the intestinal mucosa and this activation is dependent on LF binding to the intestinal epithelia (Debbabi H et al., Bovine lactoferrin induces both mucosal and system immune responses in mice. J Dairy Res 65:283-93, 1998). Later studies have demonstrated that LF potentiates thymidine incorporation into rat crypt cell DNA (Nichols B L et al., Iron is not required in the lactoferrin stimulation of thymidine incorporation into the DNA of rat crypt enterocytes. Pediatr Res 27:525-8, 1990). This trophic effect may also contribute to cell regeneration and tissue repair of intestinal mucosa in conditions such as gastroenteritis. Anabolic effects of orally administered bovine LF on visceral organ growth and protein synthesis have been evaluated in an unsuckled newborn piglet model. Animals (n=18) were randomly assigned to one of three dietary treatment groups: i) formula alone (10 mL/h), ii) formula with physiologic levels (1 mg/mL) of bovine LF or iii) formula with colostrum. After 24 h of feeding, hepatic protein synthesis in animals fed either formula containing LF or colostrum was similar and was significantly higher than the formula alone control group. These results indicate that feeding of LF-supplemented formula increases hepatic protein synthesis in the newborn, suggesting an anabolic function for LF in the neonates (Burrin D G et al., Orally administered lactoferrin increases hepatic protein synthesis in formula-fed newborn pigs. Pediatr Res 40:72-6, 1996).
Lactoferrin's interaction with neutrophils and mononuclear phagocytes is potentially of great importance in a variety of inflammatory processes. When given orally (10 mg×5 doses on alternate days), LF inhibits carrageenan-induced inflammation by 40-50%. This inhibition is associated with a substantial decrease in IL-6 production by splenocytes and LPS-induced TNF-α production. The decreased ability of spleen cells to produce inflammatory cytokines in the LF-treated group indicates that hyporeactivity of the immune system cells may be the basis for the inhibition of carrageenan-induced inflammation (Zimecki M et al., Oral treatment of rats with bovine lactoferrin inhibits carrageenan-induced inflammation: Correlation with decreased cytokine production. Arch Immunol Ther Exp Warsz 46:361-5, 1998).
Removal of endotoxins and cholesterol from the GI tract is another function of LF. Bovine LF is protective against lethal shock induced by intravenously administered endotoxin as evaluated in a germ-free, colostrum-deprived, immunologically ‘virgin’ piglet model. Pre-feeding with LF resulted in a significant decrease in piglet mortality compared to control animals (16.7% mortality in LF-treated animals versus 73.7% mortality in controls) (Lee W J et al., The protective effects of lactoferrin feeding against endotoxin lethal shock in germfree piglets. Infect Immun 66:1421-6, 1998). Lactoferrin-mediated protection against endotoxin challenge was also correlated with both resistance to induction of hypothermia and an overall increase in wellness. In vitro studies using a flow cytometric assay system demonstrated that endotoxin binding to porcine monocytes was inhibited by LF in a dose-dependent fashion, suggesting that the mechanism of LF action in vivo could be possibly due to the prevention of induction of monocyte/macrophage-derived inflammatory-toxic cytokines.
Oral administration of bovine LF with milk suppressed the proliferation of intestinal clostridium species and fecal excretion of anaerobic pathogens (Teraguchi S et al., Bacteriostatic effect of orally administered bovine lactoferrin on proliferation of Clostridium species in the gut of mice fed bovine milk. Appl Environ Microbiol 61:501-6, 1995). Furthermore, supplementation of milk with bovine LF also suppresses bacterial translocation, mainly members of the family Enterobacteriaceae, from the intestines to the mesenteric lymph nodes (Teraguchi S et al., Orally administered bovine lactoferrin inhibits bacterial translocation in mice fed bovine milk. Appl Environ Microbiol 61:4131-4, 1995). Oral administration of a solution of 1% bovine LF for three to four weeks decreased Helicobacter pylori counts in the stomach by 10% and also exerted a potent inhibitory effect on the gut attachment of the bacterium. This resulted in a marked decline in the serum antibody titer against H. pylori to an undetectable level (Wada T et al., The therapeutic effect of bovine lactoferrin in the host infected with Helicobacter pylori. Scand J Gastroenterol 34:238-43, 1999). Prophylactic and therapeutic effects of oral dosages of LF against intractable stomatitis in vivo have also been reported (Sato N et al., Lactoferrin inhibits Bacillus cereus growth and heme analogs recover its growth. Biol Pharm Bull 22:197-9, 1999).
Certain bioactive components of milk have prebiotic activity and promote growth of beneficial bacteria such as Lactobacillus spp. and Bifidobacterium spp. in vivo. It is known that the large intestine of breast-fed infants is colonized predominantly by Bifidobacterium spp., which have protective effects against enteric pathogens. Earlier studies suggested that LF derived from human and bovine sources of mature milk enhances the growth of B. infantis, B. breve and B. bifidum in vitro, in a dose-dependent manner (Roberts A K et al., Supplementation of an adapted formula with bovine lactoferrin—effects on the infant faecal flora. Acta Paediatr 81:119-24, 1992). Feeding trials with infant formula with LF supplementation (100 mg/mL) established bifidus flora in 50% of the infants at age three months. Certain peptide domains on LF have been identified to stimulate growth of Bifidobacterium spp. in vivo (Petschow B W et al., Ability of lactoferrin to promote the growth of Bifidobacterium spp. in vitro is independent of receptor binding capacity and iron saturation level. J Med Microbiol 48:541-9, 1999; Liepke C et al., Human milk provides peptides highly stimulating the growth of bifidobacteria. Eur. J. Biochem. 269:712-8, 2002; Lonnerdal B, Nutritional and physiologic significance of human milk proteins. Am J Clin Nutr 77:1537S-153S 2003).
Thus, the combination of prebiotic activity with multifunctional activities described herein makes LF a powerful nutraceutical for regulating the microbial balance, mucosal defense, nutrient absorption and healthy maintenance of the gastrointestinal tract.
Oral administration of LF, and its role as a multifunctional delivery system in the gastrointestinal tract, is clearly established in research laboratories and in several experimental trials worldwide. However, to further any commercial exploitation of LF as a prebiotic system for human health application requires an innovative technology compatible with large-scale manufacturing practices. Such technology transfer must ensure the highest standards of product safety, quality assurance and delivery of an optimal dosage for an effective functional outcome. There are four major issues critical for the commercialization of LF as a prebiotic in vivo delivery system including bioactivity, microbiological quality, endotoxin content and dosage.
First we will address the issue of bioavailability. Like most multifunctional proteins, LF activity is highly dependent on the three-dimensional or tertiary structure of the molecule. Thus, the stability of LF protein could limit its usefulness. Conditions such as the presence of metals (iron, in particular), anionic ions, salts, pH, temperature and conductivity are known to affect the functional properties of LF. Furthermore, protein isolation and processing conditions including storage, freezing/thawing, and spray-drying could also adversely affect LF bioactivity. Degradation of native LF into peptic fragments as well as the co-elution of impurities from raw material could further compromise LF applications. Therefore, LF could denature or inactivate to partially or totally lose its functional properties during large-scale manufacturing.
U.S. Pat. No. 6,172,040 to Naidu teaches certain immobilized forms of LF and their antimicrobial applications. The immobilization method disclosed in the Naidu patent relates to an ex vivo orientation of the LF molecule similar to its molecular configuration when bound to mammalian mucosa, as well as to an increased structural stability of the LF protein and to an optimal neutralization of cationic peptides to eliminate undesirable non-specific bactericidal effects. Furthermore, this immobilization step, in combination with certain formulation conditions, could potentiate LF into a powerful antibiotic that serves as an excellent system to protect against harmful pathogens. However, immobilized lactoferrin is neither designed nor is reported to have prebiotic activity.
The microbiological quality of LF starting raw materials could significantly compromise the human health applications of commercial LF. Several factors including the source of raw starting material, protein separation and harvesting methods, and manufacturing environment and storage conditions all cumulatively contribute to the LF bio-burden. Accordingly, when used as a raw starting material, whey or milk serum has the potential to carry-through fermentative streptococci (Streptococcus thermophilus, in particular) and in an acid environment could selectively enrich several yeast and molds. These microbial populations are commonly known to proliferate and competitively limit several strains of probiotic bacteria. Lactoferrin derived directly from the milk source could minimize the above problem, however contamination of the milk pool from cows with bovine mastitis could introduce several Gram-positive cocci such as Strep. uberis, Staphylococcus aureus and coagulase-negative staphylococci. Additionally, environmental contaminants such as spore-forming Bacillus spp., Acinetobacter calcoaceticus, Klebsiella oxytoca, Pseudomonas spp., and coliforms including Escherichia coli could gain entry into LF material through elution buffers, biofouled equipment and air ducts. Similarly, microbiological quality issues also exist for genetically modified organism (GMO)-derived and recombinant LFs from various expression sources such as rice, tobacco, yeast, cell cultures or transgenic animals. Therefore, elimination or significant reduction of such microbial contaminants is highly critical for human health applications of commercially available LF and for developing prebiotic delivery systems.
The endotoxin content of LF starting raw material could also adversely affect its human health applications. Lipopolysaccharides (LPS) are the outer membrane components of Gram-negative bacteria that typically consist of hydrophobic domain known as lipid-A (or endotoxin), a non-repeating core oligosaccharide, and a distal polysaccharide (or O-antigen). Endotoxins stimulate the production of cytokines and other mediators of inflammation, which in turn trigger a broad range of adverse physiological responses. Experimental evidence suggests that reactive oxygen species are important mediators of cellular injury during endotoxemia, either as a result of macromolecular damage or by interfering with extracellular and intracellular regulatory processes. In addition, nitric oxide is thought to play a key role in the pathogenesis of endotoxic shock. The Gram-negative bio-burden of milk or its derivatives used in the LF isolation, processing plant environment and conditions cumulatively contribute to the endotoxin levels in LF material. The potential sources of endotoxin contamination during isolation of protein materials have been recently reviewed (Majde J A, Microbial cell-wall contaminants in peptides—a potential source of physiological artifacts. Peptides 14:629-32, 1993). Additionally, Rylander (Rylander R, Endotoxin in the Environment—exposure and effects. J Endotoxin Res 8:241-52, 2002) has reviewed the occurrence of endotoxin in different environmental conditions and further pointed out the risks associated with non-bacterial endotoxins, particularly 1,3-β-D-glucan from mold cell walls. Thus, the microbiological standards of chromatographic resins, sanitation practices of processing equipment and even more significantly the water quality used in LF isolation, could cumulatively contribute to the endotoxin levels of the isolated LF material and thereby could limit the in vivo applications of commercial LF.
Since LF is a multifunctional protein with a defining role in various physiological pathways, its activity is highly dependant on dosage and a proper delivery system. Regulatory proteins are like traffic signals and thus at optimal dosages function positively in a beneficial manner promoting a physiological function, while at other dosages (usually high concentrations) function negatively through feedback inhibition by blocking body functions. In order to maintain an optimum physiological balance, LF is cleared by liver and spleen at a catabolic rate of 5.7 mg/day (Bennett R M, Kokocinski T, Lactoferrin turnover in man. Clin Sci 57:453-60, 1979).
Lactoferrin co-exists with an array of molecules in different mucosal secretions under varying environmental or physiologic conditions. These substrates and/or physio-chemical conditions exert a specific effect on the structural reorganization of LF molecule and thereby define its multifunctional properties. In humans, the normal levels of LF are 1-2 mg/mL in breast milk, tears and gastric mucins, 0.1-1 mg/mL in saliva, crevicular fluids, and sperm, <0.01 mg/mL in synovial fluids and plasma and the secondary granules of neutrophils contain about 0.01 mg/106 cells. However, these levels significantly rise by 10- to 100-fold during infections such as mastitis and parotitis (Tabak L et al., Changes in lactoferrin and other proteins in a case of recurrent parotitis. J Oral Pathol 1:97-9, 1978; Harmon R J et al., Changes in lactoferrin, immunoglobulin G, bovine serum albumin, and α-lactalbumin during acute experimental and natural coliform mastitis in cows. Infect Immun 13:533-42, 1976). In such abnormal conditions, LF aggregates into dimeric and tetrameric complexes that could subsequently lead to LF dysfunctionality. (Bennett R M et al., Calcium-dependant polymerization of lactoferrin. Biochem Biophys Res Commun 101:88-95, 1981).
Lactoferrin dosage, therefore, is highly critical in the development of any in vivo delivery system. Furthermore, in the design of such a therapeutic, estimation of average daily intake (ADI) values for LF in the human plays a significant role. According to the United States Department of Agriculture (USDA) Continuing Survey of Food Intakes by Individuals (CSFII) data from 1994-96, the average intake of milk and milk products on both a gram per day (g/d) and gram per kilogram of body weight per day (g/kg bw/d) have been calculated. The CSFII 1994-96 data is based on dietary information from individuals of all ages collected between January 1994 and January 1997. Considering that cow's milk contains 0.1 mg/mL to 0.2 mg/mL of LF, on an average, children 1 to 12 years old and teens 13 to 19 years old consume about 396 g milk/day and 377 g milk/day, respectively. This is equivalent to 38 to 40 mg LF/day. Adults (20+) consume less milk, 240 g/d and their intake of LF is equal to about 24 mg/day. The consumption of LF for milk consumers in the 90th percentile averages 73 mg/d for children, 75 mg/d for teens and 50 mg/d for adults.